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Abstract

Kawasaki disease (KD) is an acute vasculitis in children that can cause coronary artery abnormalities. Its diagnosis is challenging, and many cytokines, chemokines, acute phase reactants, and growth factors have failed evaluation as specific biomarkers to distinguish KD from other febrile illnesses. We performed protein profiling, comparing plasma from children with KD with febrile control (FC) subjects to determine if there were specific proteins or peptides that could distinguish the two clinical states.Plasma from three independent cohorts from the blood of 68 KD and 61 FC subjects was fractionated by anion exchange chromatography, followed by surface-enhanced laser desorption ionization (SELDI) mass spectrometry of the fractions. The mass spectra of KD and FC plasma samples were analyzed for peaks that were statistically significantly different.A mass spectrometry peak with a mass of 7,860 Da had high intensity in acute KD subjects compared to subacute KD (p = 0.0003) and FC (p = 7.9 x 10-10) subjects. We identified this peak as a novel truncated form of serum amyloid A with N-terminal at Lys-34 of the circulating form and validated its identity using a hybrid mass spectrum immunoassay technique. The truncated form of serum amyloid A was present in plasma of KD subjects when blood was collected in tubes containing protease inhibitors. This peak disappeared when the patients were examined after their symptoms resolved. Intensities of this peptide did not correlate with KD-associated laboratory values or with other mass spectrum peaks from the plasma of these KD subjects.Using SELDI mass spectrometry, we have discovered a novel truncated form of serum amyloid A that is elevated in the plasma of KD when compared with FC subjects. Future studies will evaluate its relevance as a diagnostic biomarker and its potential role in the pathophysiology of KD.

Abstract

To get a better understanding of the ongoing in situ environmental changes preceding the brain tumorigenesis, we assessed cerebrospinal fluid (CSF) proteome profile changes in a glioma rat model in which brain tumor invariably developed after a single in utero exposure to the neurocarcinogen ethylnitrosourea (ENU). Computationally, the CSF proteome profile dynamics during the tumorigenesis can be modeled as non-smooth or even abrupt state changes. Such brain tumor environment transition analysis, correlating the CSF composition changes with the development of early cellular hyperplasia, can reveal the pathogenesis process at network level during a time before the image detection of the tumors. In our controlled rat model study, matched ENU- and saline-exposed rats' CSF proteomics changes were quantified at approximately 30, 60, 90, 120, 150days of age (P30, P60, P90, P120, P150). We applied our transition-based network entropy (TNE) method to compute the CSF proteome changes in the ENU rat model and test the hypothesis of the critical transition state prior to impending hyperplasia. Our analysis identified a dynamic driver network (DDN) of CSF proteins related with the emerging tumorigenesis progressing from the non-hyperplasia state. The DDN associated leading network CSF proteins can allow the early detection of such dynamics before the catastrophic shift to the clear clinical landmarks in gliomas. Future characterization of the critical transition state (P60) during the brain tumor progression may reveal the underlying pathophysiology to device novel therapeutics preventing tumor formation. More detailed method and information are accessible through our website at http://translationalmedicine.stanford.edu.

Abstract

Biomarker discovery datasets created using mass spectrum protein profiling of complex mixtures of proteins contain many peaks that represent the same protein with different charge states. Correlated variables such as these can confound the statistical analyses of proteomic data. Previously we developed an algorithm that clustered mass spectrum peaks that were biologically or technically correlated. Here we demonstrate an algorithm that clusters correlated technical aliases only.In this paper, we propose a preprocessing algorithm that can be used for grouping technical aliases in mass spectrometry protein profiling data. The stringency of the variance allowed for clustering is customizable, thereby affecting the number of peaks that are clustered. Subsequent analysis of the clusters, instead of individual peaks, helps reduce difficulties associated with technically-correlated data, and can aid more efficient biomarker identification.This software can be used to pre-process and thereby decrease the complexity of protein profiling proteomics data, thus simplifying the subsequent analysis of biomarkers by decreasing the number of tests. The software is also a practical tool for identifying which features to investigate further by purification, identification and confirmation.

Abstract

Understanding the early relationship between brain tumor cells and their environment could lead to more sensitive biomarkers and new therapeutic strategies. We have been using a rodent model of neurocarcinogenesis in which all animals develop brain tumors by six months of age to establish two early landmarks in glioma development: the appearance of a nestin(+) cell at thirty days of age and the appearance of cellular hyperplasia between 60 and 120 days of age. We now report an assessment of the CSF proteome to determine the changes in protein composition that occur during this period.Nestin(+) cell clusters and microtumors were assessed in 63 ethylnitrosourea-exposed rats on 30, 60, and 90 days of age. CSF was obtained from the cisterna magna from 101 exposed and control rats at 30, 60, and 90 days and then analyzed using mass spectrometry. Differentially expressed peaks were isolated and identified.Nestin(+) cells were noted in all ethylnitrosourea-exposed rats assessed pathologically. Small microtumors were noted in 0%, 18%, and 67% of 30-, 60-, and 90-day old rats, respectively (p<0.05, Chi square). False Discovery Rate analysis of peak intensities showed that the number of true discoveries with p<0.05 increased markedly with increasing age. Isolation and identification of highly differentially detected proteins at 90 days of age revealed increases in albumin and a fragment of ?1 macroglobulin and alterations in glutathionylated transthyretin.The presence of increased albumin, fragments of cerebrospinal fluid proteins, and glutathione breakdown in temporal association with the development of cellular hyperplasia, suggests that, similar to many other systemic cancers, inflammation and oxidative stress is playing an important early role in the host's response to brain tumor development and may be involved in affecting the early growth of brain tumor.

Abstract

We sought to investigate protein biomarkers for stress urinary incontinence (SUI) in vaginal tissues using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and examine if this is a reliable methodology to examine proteins in small tissue specimens.? We compared protein expression profile of vaginal tissue from women with SUI and continent controls. A 22.6kDa peak was identified by subsequent weak cation-exchange, reverse-phase fractionation, gel electrophoresis, and trypsin digestion, then analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and MALDI MS-MS. Biomarker identity and expression level were confirmed by Western-blotting and immunohistochemistry.Expression of the 22.6kDa protein, identified as SM-22?, was significantly higher in women with SUI versus controls. A 3×3-mm tissue sample was sufficient for identification. Western-blot/immunohistochemistry confirmed the SELDI-TOS MS findings.SM-22?, a marker for myofibroblasts, was identified as a biomarker of SUI. Differential protein profiling by SELDI-TOF MS is a powerful and reliable tool for urogynecological research as it allows us to study an array of proteins simultaneously using small tissue samples.

Abstract

Kawasaki disease is an acute vasculitis of infants and young children that is recognized through a constellation of clinical signs that can mimic other benign conditions of childhood. The etiology remains unknown and there is no specific laboratory-based test to identify patients with Kawasaki disease. Treatment to prevent the complication of coronary artery aneurysms is most effective if administered early in the course of the illness. We sought to develop a diagnostic algorithm to help clinicians distinguish Kawasaki disease patients from febrile controls to allow timely initiation of treatment.Urine peptidome profiling and whole blood cell type-specific gene expression analyses were integrated with clinical multivariate analysis to improve differentiation of Kawasaki disease subjects from febrile controls.Comparative analyses of multidimensional protein identification using 23 pooled Kawasaki disease and 23 pooled febrile control urine peptide samples revealed 139 candidate markers, of which 13 were confirmed (area under the receiver operating characteristic curve (ROC AUC 0.919)) in an independent cohort of 30 Kawasaki disease and 30 febrile control urine peptidomes. Cell type-specific analysis of microarrays (csSAM) on 26 Kawasaki disease and 13 febrile control whole blood samples revealed a 32-lymphocyte-specific-gene panel (ROC AUC 0.969). The integration of the urine/blood based biomarker panels and a multivariate analysis of 7 clinical parameters (ROC AUC 0.803) effectively stratified 441 Kawasaki disease and 342 febrile control subjects to diagnose Kawasaki disease.A hybrid approach using a multi-step diagnostic algorithm integrating both clinical and molecular findings was successful in differentiating children with acute Kawasaki disease from febrile controls.

Abstract

Glutathione peroxidase-3 (Gpx3), also known as plasma or extracellular glutathione peroxidase, is a selenoprotein secreted primarily by kidney proximal convoluted tubule cells. In this study Gpx3(-/-) mice have been produced and immunocytochemical techniques have been developed to investigate Gpx3 metabolism. Gpx3(-/-) mice maintained the same whole-body content and urinary excretion of selenium as did Gpx3(+/+) mice. They tolerated selenium deficiency without observable ill effects. The simultaneous knockout of Gpx3 and selenoprotein P revealed that these two selenoproteins account for >97% of plasma selenium. Immunocytochemistry experiments demonstrated that Gpx3 binds selectively, both in vivo and in vitro, to basement membranes of renal cortical proximal and distal convoluted tubules. Based on calculations using selenium content, the kidney pool of Gpx3 is over twice as large as the plasma pool. These data indicate that Gpx3 does not serve in the regulation of selenium metabolism. The specific binding of a large pool of Gpx3 to basement membranes in the kidney cortex strongly suggests a need for glutathione peroxidase activity in the cortical peritubular space.

Abstract

Our objective was to compare protein profiles of cerebrospinal fluid between control animals and those subjected to cardiopulmonary bypass after moderate versus deep hypothermic circulatory arrest with selective cerebral perfusion.Immature Yorkshire piglets were assigned to one of four study groups: (1) deep hypothermic circulatory arrest at 18 degrees C, (2) deep hypothermic circulatory arrest at 18 degrees C with selective cerebral perfusion, (3) moderate hypothermic circulatory arrest at 25 degrees C with selective cerebral perfusion, or (4) age-matched control animals without surgery. Animals undergoing cardiopulmonary bypass were cooled to their assigned group temperature and exposed to 1 hour of hypothermic circulatory arrest. After arrest, animals were rewarmed, weaned off bypass, and allowed to recover for 4 hours. Cerebrospinal fluid collected from surgical animals after the recovery period was compared with cerebrospinal fluid from controls by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Protein spectra were analyzed for differences between groups by Mann-Whitney U test and false discovery rate analysis.Baseline and postbypass physiologic parameters were similar in all surgical groups. A total of 194 protein peaks were detected. Compared with controls, groups 1, 2, and 3 had 64, 100, and 13 peaks that were significantly different, respectively (P < .05). Three of these peaks were present in all three groups. Cerebrospinal fluid protein profiles in animals undergoing cardiopulmonary bypass with moderate hypothermic circulatory arrest (group 3) were more similar to controls than either of the groups subjected to deep hypothermia.The mass spectra of cerebrospinal fluid proteins are altered in piglets exposed to cardiopulmonary bypass and hypothermic circulatory arrest. Moderate hypothermic circulatory arrest (25 degrees C) with selective cerebral perfusion compared with deep hypothermic circulatory arrest (18 degrees C) is associated with fewer changes in cerebrospinal fluid proteins, when compared with nonbypass controls.

Abstract

Preterm labor (PTL) is frequently associated with inflammation. We hypothesized that biomarkers during pregnancy can identify pregnancies most at risk for development of PTL. An inflammation-induced mouse model of PTL was used. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to analyze and compare the plasma protein (PP) profile between CD-1 mice injected intrauterine with either lipopolysaccharide (LPS) or PBS on d 14.5 of gestation. The median differences of normalized PP peaks between the two groups were determined using the Mann-Whitney U test and the false discovery rate. In a second series of experiments, both groups of mice were injected with a lower dose of LPS. A total of 1665 peaks were detected. Thirty peaks were highly differentially expressed (p < 0.0001) between the groups. Two 11 kDa protein peaks were identified by MALDI-TOF/TOF-MS and confirmed to be mouse serum amyloid A (SAA) 1 and 2. Plasma SAA2 levels were increased in LPS-treated animals compared with controls and in LPS-treated animals that delivered preterm vs. those that delivered at term. SAA2 has the potential to be a plasma biomarker that can identify pregnancies at risk for development of PTL.

Abstract

Alpha-zirconium phosphate nanoplatelets (alpha-ZrPN) were studied as a binding agent for phosphopeptides. Nanoplatelets of alpha-zirconium phosphate were incubated overnight with zirconium oxychloride, followed by centrifugation, and washed twice with water followed by an aqueous solution of 80% acetonitrile to form the binding agent. Alpha-ZrPN were able specifically to capture phosphoserine-containing peptides from a tryptic digest of a complex peptide mixture in which its abundance was only 0.05%. Alpha-ZrPN also bound peptides containing phosphothreonine and phosphotyrosine. The limit of detection for phosphopeptides is approximately 2 fmol, based on using matrix-assisted laser desorption/ionization mass spectrometry. Alpha-ZrPN were applied for the analysis of tryptic digests of mouse liver and leukemia cell phosphoproteomes and succeeded in identifying 158 phosphopeptides (209 phosphorylation sites) from 101 phosphoproteins in mouse liver lysate and 78 phosphopeptides (104 phosphorylation sites) from 59 phosphoproteins in leukemia cell extract. For these two tryptic digests, the alpha-ZrPN approach is able to capture more phosphopeptides than that obtained from TiO2 particles or from Fe(3+)-IMAC beads, but each method is able to bind some phosphopeptides that the others do not.

Abstract

Sulfur is essential for life, with important roles in biological structure and function. However, because of a lack of suitable biophysical techniques, in situ information about sulfur biochemistry is generally difficult to obtain. Here, we present an in situ sulfur X-ray absorption spectroscopy (S-XAS) study of living cell cultures of the mammalian renal epithelial MDCK cell line. A great deal of information is retrieved from a characteristic sulfonate feature in the X-ray absorption spectrum of the cell cultures, which can be related to the amino acid taurine. We followed the time and dose dependence of uptake of taurine into MDCK cell monolayers. The corresponding uptake curves showed a typical saturation behavior with considerable levels of taurine accumulation inside the cells (as much as 40% of total cellular sulfur). We also investigated the polarity of uptake of taurine into MDCK cells, and our results confirmed that uptake in situ is predominantly a function of the basolateral cell surface.

Abstract

Preterm infants are at risk of developing sepsis, necrotizing enterocolitis (NEC), chronic lung disease (CLD), and retinopathy of prematurity (ROP). We used high-throughput mass spectrometry to investigate whether cord blood proteins can be used to predict development of these morbidities. Cord blood plasma from 44 infants with a birth weight of <1500 g was analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF). Six infants developed ROP >or=stage II, 10 CLD, three sepsis, and one NEC. We detected 814 protein signals representing 330 distinct protein species. Nineteen biomarkers were associated with development of >or=stage II ROP [false-discovery rate (FDR) <5%] and none with CLD. Several proteins with molecular weight (Mr) 15-16 kD and pI 4-5 were detected with increased abundance in infants with ROP, while similar Mr proteins with pI 7-9 were less abundant in these patients. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sequence analysis identified these proteins as alpha-, beta-, and gamma-globin chains. Partial deamidation of Asn139 in beta-globin chains was observed only in the pI 4-5 proteins. We conclude that there are several promising biomarkers for the risk of ROP. Deamidation of globin chains is especially promising and may indicate underlying prenatal pathologic mechanisms in ROP. Validation studies will be undertaken to determine their clinical utility.

Abstract

Discovering valid biological information from surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) depends on clear experimental design, meticulous sample handling, and sophisticated data processing. Most published literature deals with the biological aspects of these experiments, or with computer-learning algorithms to locate sets of classifying biomarkers. The process of locating and measuring proteins across spectra has received less attention. This process should be tunable between sensitivity and false-discovery, and should guarantee that features are biologically meaningful in that they represent chemical species that can be identified and investigated. Existing feature detection in SELDI-TOF MS is not optimal for acquiring biologically relevant data. Most methods have so many user-defined settings that reproducibility and comparability among studies suffer considerably. To address these issues, we have developed an approach, called simultaneous spectrum analysis (SSA), which (i) locates proteins across spectra, (ii) measures their abundance, (iii) subtracts baseline, (iv) excludes irreproducible measurements, and (v) computes normalization factors for comparing spectra. SSA uses only two key parameters for feature detection and one parameter each for quality thresholds on spectra and peaks. The effectiveness of SSA is demonstrated by identifying proteins differentially expressed in SELDI-TOF spectra from plasma of wild-type and knockout mice for plasma glutathione peroxidase. Comparing analyses by SSA and CiphergenExpress Data Manager 2.1 finds similar results for large signal peaks, but SSA improves the number and quality of differences betweens groups among lower signal peaks. SSA is also less likely to introduce systematic bias when normalizing spectra.

Abstract

Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors. Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation. Here we show that Ca2+ and K+ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death. The Ca2+ channel blockers, nickel and econazole, and the K+ channel blockers, tetraethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca2+ ([Ca2+](i)), the decrease in intracellular K+, and apoptosis. Thapsigargin, which releases Ca2+ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in [Ca2+](i) in Jurkat cells, indicating that the initial increase in [Ca2+](i) is from intracellular stores. The rise in [Ca2+](i) precedes a decrease in intracellular K+, and elevated extracellular K+ prevents granulysin-mediated cell death. In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated. Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.

Abstract

Individuals supplemented with selenium have reduced incidence of prostate cancer. This study determines whether selenomethionine specifically affects the secretion of prostate specific antigen (PSA) in vitro.LNCaP cells were supplemented with selenomethionine for 7 days. PSA secretion was determined by ELISA. Cell proliferation was assessed by enumeration of trypan blue excluding cells. Colony formation was determined in soft agar. Cell cycle distribution was determined by FACS analysis of propidium iodide stained cells.Selenomethionine at > or = 70 microM inhibited LNCaP cell growth and colony formation. 0-100 microM selenomethionine did not affect the secretion of PSA by LNCaP cells in cell culture supernatants when normalized to the number of cells in culture. At supra-nutritional concentrations of selenomethionine, LNCaP cells had longer G(0)/G(1) phase in agreement with the inhibitory effects on cell growth.PSA secretion is not specifically inhibited by concentrations of selenomethionine corresponding to plasma selenium concentrations found in individuals supplemented with chemopreventive concentrations of selenized yeast. These data suggest that changes in serum PSA levels in individual patients during selenium supplementation is not an effect specific for PSA secretion, but rather may be a useful indicator for changes in disease progression in individual patients.

Abstract

Extracellular glutathione peroxidase (eGPx) is a secreted selenoenzyme with GPx activity. eGPx protein and activity are found in blood plasma and other extracellular fluids. eGPx in plasma is predominantly derived from the proximal tubules of kidneys in humans. Two types of human proximal tubule cells were cultured on semipermeable polycarbonate membranes to determine whether these cells secrete eGPx in a polarized direction. Immortalized human proximal tubule HK-2 cells and primary human proximal tubule cells formed confluent monolayers when cultured on these membrane inserts in culture dishes, as evidenced by transepithelial resistance. Both cell lines also constituted a barrier to diffusion of a fluoresceinated dextran of 75 kDa, a size similar to eGPx homotetramers. In both cell lines, 6- to 12-fold more 35S-methionine-labeled eGPx was immunoprecipitated from the basolateral media than from the apical media, indicating basolateral secretion of eGPx. eGPx was immunolocalized to the extracellular fluid at the basolateral surface of proximal tubules in human kidney. These data support the conclusion that eGPx is secreted through the basolateral membrane of human kidney proximal tubule cells into the extracellular fluid of the kidney, and from there enters blood plasma.

Abstract

Extracellular glutathione peroxidase (E-GPx) is a selenoenzyme that reduces hydrogen peroxide and organic peroxides. All plasma glutathione peroxidase (GPx) activity in humans is attributable to E-GPx. The gastrointestinal (GI) tract also synthesizes and secretes E-GPx into the extracellular milieu. Endogenously generated oxidants have been implicated in inflammatory bowel disease (IBD). We evaluated E-GPx levels in a mouse model of IBD using dextran sodium sulfate (DSS). Histologic lesions of the lower GI tract consisted of multifocal areas of mucosal erosion denuded of epithelial cells, reduction in goblet cells, dilated crypts, crypt collapse, submucosal edema, and transmural distribution of mixed inflammatory infiltrates. On d 7, plasma GPx activity in the DSS group increased by 61% compared with the control group (p < 0.05). Western blot analysis demonstrated a 64% increase in E-GPx protein in the plasma of the DSS group after 7 d of treatment (p < 0.01). As the major source of plasma GPx is the kidney, we determined whether the increase in plasma GPx activity and protein was caused by a change in E-GPx synthesis by the kidney. After 3 and 7 d of DSS treatment, E-GPx mRNA levels, relative to glyceraldehyde-3-phosphate dehydrogenase, increased in the kidney (p < 0.05) without a concomitant increase in cellular GPx mRNA on d 7. These results suggest that the inflammatory injury in the intestine elicits an increase in E-GPx in the plasma that is associated with an increase in E-GPx mRNA in the kidney. This implies that renal production of E-GPx may be sensitive to insults distal to the kidney.

Abstract

Exposure to ozone injures respiratory epithelium, and the mechanisms may involve the generation of reactive oxygen species (ROS). This study tested the hypothesis that ozone exposure increases the airway burden of ROS to a greater degree in smokers than nonsmokers, and that this effect is independent of ozone-induced changes in spirometry. Healthy subjects were selected as either responders (decrement in FEV1 > 15%) or nonresponders (decrement in FEV1 < 5%) to ozone; each underwent 2 exposures to ozone and 1 to air, with bronchoalveolar lavage (BAL) performed 30 min (early) and 18 h (late) after exposure. Release of superoxide anion (O2(-)) was used as a measure of ROS release by all BAL cells, and flow cytometry was used to detect ROS production in alveolar macrophages (AM) only. Recovery of AM was approximately threefold greater in smokers than nonsmokers. Unstimulated, but not stimulated, cells obtained by BAL from smokers released approximately twofold greater amounts of O2(-) than cells from nonsmokers, both early and late after ozone exposure (p =.012 and p =.046, respectively). Stimulated, but not unstimulated, ROS generation by AM from smokers increased following ozone exposure, but the ozone effect was not significant. ROS production by AM decreased in nonsmokers (air vs. ozone late, p =.014). Total protein, albumin, and immunoglobulin M (IgM) increased in BAL fluid, consistent with an increase in epithelial permeability. In addition, the concentration of alpha2-macroglobulin increased approximately threefold 18 h after exposure in nonsmokers (p

Abstract

Selenium (Se) in selenite is present in an oxidized state, and must be reduced for it to be incorporated as selenocysteine into selenoenzymes such as glutathione peroxidase (GPx). In vitro, Se, as in selenite, can be reduced utilizing glutathione (GSH) and glutathione reductase (GRed). We determined the effects of decreasing GSH levels, inhibiting GRed activity, and decreasing cellular NADPH on the selenite-dependent rate of GPx synthesis in cultured cells: PC3, CHO, and the E89 glucose-6-phosphate dehydrogenase (G-6-PD)-deficient cell line. A novel statistical analysis method was developed (using Box Cox transformed regression and a bootstrap method) in order to assess the effects of these manipulations singly and in combinations. Buthionine sulfoximine (BSO) was used to decrease GSH levels, 1,3 bis-(2 chloroethyl)-1 -nitrosourea (BCNU) was used to inhibit GRed activity and methylene blue (MB) was used to decrease cellular NADPH levels. This statistical method evaluates the effects of BSO, BCNU, MB and selenite alone and in combinations on GPx activity. Decreasing the GSH level (< 5% of control) did not have an effect on the selenite-dependent rate of GPx synthesis in PC3 or CHO cells, but did have a small inhibitory effect on the rate of GPx synthesis in E89 cells. Inhibiting GRed activity was also associated with either no effect (CHO, E89) or a small effect (PC3) on GPx activity. In contrast, decreasing NADPH levels in cells treated with MB was associated with a large decrease in the selenite-dependent rate of GPx synthesis to 36, 34 and 25% of control in PC3, CHO, and E89 cells, respectively. The effects of BSO plus BCNU were not synergistic in any of the cell lines. The effects of BSO plus MB were synergistic in G-6-PD-deficient E89 cells, but not in PC3 or CHO cells. We therefore conclude that under normal culture conditions, NADPH, and not glutathione, is the primary reductant of Se in selenite to forms that are eventually incorporated into GPx. For cells with abnormal ability to generate NADPH, lowering the GSH levels had a small effect on selenite-dependent GPx synthesis. GRed activity is not required for the selenite-dependent synthesis of GPx.

Abstract

Extracellular glutathione peroxidase (E-GPx) is a selenium-dependent enzyme that can reduce hydrogen peroxide and phospholipid hydroperoxides. E-GPx is found in plasma and extracellular fluids such as bronchoalveolar lavage fluid. Because lung is one of the tissues that is capable of synthesizing and secreting E-GPx, the effect of exposure to hyperoxia on E-GPx in plasma and lung were studied in an injury model of hyperoxia exposure in adult mice. Exposure to 100% oxygen for 72 h resulted in an increase of 55% in plasma GPx activity and an increase of 50% in the amount of E-GPx protein in the plasma. Exposure to hyperoxia was also associated with an increase in the amount of E-GPx protein in lungs. The 7-fold increase in the amount of E-GPx protein in lungs was not due to plasma contamination of lungs from mice exposed to hyperoxia. E-GPx in the lung is calculated to account for 10% of lung GPx activity in control mice. However, E-GPx is calculated to account for 45% of lung GPx activity in the lungs of mice exposed to hyperoxia for 72 h. Further studies are needed to determine whether the increase in lung E-GPx is due to changes in translation or stability of E-GPx. The role of E-GPx in protecting the lung from oxidative damage warrants further study.

Abstract

Extracellular glutathione peroxidase (EGPx) is a glycosylated selenoprotein capable of reducing hydrogen peroxide, organic hydroperoxides, free fatty acid hydroperoxides, and phosphatidylcholine hydroperoxides. We found that human large intestinal explant cultures synthesize EGPx and cellular glutathione peroxidase (CGPx) and secrete EGPx. The level of EGPx mRNA expression relative to alpha-tubulin was similar throughout the mouse gastrointestinal tract. EGPx mRNA transcripts have been localized to mature absorptive epithelial cells in human and mouse large intestine. Western blot analysis of mouse intestinal protein has demonstrated the presence of EGPx protein in the small intestine, cecum, and large intestine, with the highest protein levels found in the cecum. Immunohistochemistry studies of human large intestine and mouse small and large intestine sections demonstrated the presence of EGPx protein within mature absorptive epithelial cells. In human large intestine and mouse small intestine, EGPx protein is also present in the extracellular milieu. These results suggest a role for EGPx in protection of the intestinal tract from peroxidative damage and/or in intercellular metabolism of peroxides.

Abstract

Selenium-dependent extracellular glutathione peroxidase (E-GPx) is found in plasma and other extracellular fluids. Previous studies have indicated that patients with chronic renal failure on dialysis have low plasma GPx activity. In this study, dialysis patients had approximately 40% of control plasma GPx activity, while anephric individuals had lowest plasma GPx activities ranging from 2 to 22% of control. The residual plasma GPx activity in anephric individuals could be completely precipitated by anti-E-GPx antibodies, indicating that all plasma GPx activity can be attributed to E-GPx in both normal and anephric individuals. Plasma GPx activity rises rapidly following kidney transplantation, often reaching normal values within 10 days. The plasma GPx activity in some transplanted patients rises to levels higher than the normal range, followed by a return to the normal range. Since E-GPx in the kidney is primarily synthesized in the proximal tubules, we investigated whether nephrotoxic agents known to disrupt proximal tubule function also affected plasma GPx activity. The beta-lactam antibiotic cephaloglycin rapidly caused a decrease in plasma GPx activity in rabbits. In addition, the chemotherapeutic agent ifosfamide caused a decrease in plasma GPx activity in pediatric osteosarcoma patients. Fanconi syndrome associated with either ifosfamide therapy or valproic acid therapy also caused a decrease in plasma GPx activity. Thus plasma GPx activity is related to kidney function and is decreased in certain situations where nephrotoxic drugs are administered. Monitoring plasma GPx activity may have predictive value in evaluating the function of transplanted kidneys or in predicting those patients particularly at risk of nephrotoxic injury associated with certain medications.

Abstract

Extracellular glutathione peroxidase (EGPx) is a secreted selenium-dependent enzyme that reduces hydroperoxides and organic hydroperoxides. Selenium deficiency in females is associated with infertility and spontaneous abortion, suggesting a role for selenium-requiring proteins during embryonic development. To gain insight into functions of EGPx in vivo, we determined sites of murine EGPx synthesis by in situ hybridization during embryogenesis and in adult tissues. At E7.5 of development, high EGPx expression was found in the maternally derived deciduum, with lower levels of accumulation in the embryonic visceral endoderm. At E9.5, the major sites of expression were the yolk sac endoderm and heart musculature. By E16.5, EGPx mRNA expression persisted in yolk sac endoderm but also accumulated significantly in atrially derived myocytes, ossification centers, adipose tissue, intestinal epithelium, and in a ventral-to-dorsal gradient in developing skin. Glutathione peroxidase activity due to EGPx protein was identified in the fluids surrounding the developing mouse embryo at midgestation. The expression of EGPx in tissues at the maternal-fetal interface--deciduum, visceral yolk sac, and skin--suggests that EGPx may serve to protect the embryo from oxidant damage. In adult mice, we identified the S1 segment of the kidney proximal tubules as the primary site of EGPx mRNA accumulation, with lower EGPx levels in atrial cardiac muscle, intestine, skin, and adipose tissue. These findings suggest that EGPx may serve a wider antioxidant role than previously recognized in the interstitium of multiple localized tissues, particularly those associated with the active transport of lipids.

Abstract

Exposure to ozone causes symptoms, changes in lung function, and airway inflammation. We studied whether individuals who differ in lung-function responsiveness to ozone, or in smoking status, also differ in susceptibility to airway inflammation. Healthy subjects were selected on the basis of responsiveness to a classifying exposure to 0.22 ppm ozone for 4 h with exercise (responders, with a decrease in FEV1 > 15%; and non-responders, with a decrease in FEV1 < 5%). Three groups were studied: nonsmoker-nonresponders (n = 12), nonsmoker-responders (n = 13), and smokers (n = 13, 11 nonresponders and two responders). Each subject underwent two exposures to ozone and one to air, separated by at least 3 wk; bronchoalveolar and nasal lavages were performed on three occasions: immediately (early) and 18 h (late) after ozone exposure, and either early or late after air exposure. Recovery of polymorphonuclear leukocytes (PMN) increased progressively in all groups, and by up to 6-fold late after ozone exposure. Interleukin-6 (IL-6) and IL-8 increased early (by up to 10-fold and up to 2-fold, respectively), and correlated with the late increase in PMN. Lymphocytes, mast cells, and eosinophils also increased late after exposure. We conclude that ozone-induced airway inflammation is independent of smoking status or airway responsiveness to ozone.

Abstract

Host protection against pneumococcal disease i primarily mediated by phagocytosis. We developed and standardized an opsonophagocytic assay using HL-60 cells (human promyelocytic leukemia cells). Fifty-five serum samples were analyzed for the presence of functional antibody against seven pneumococcal serogroups or serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by using differentiated HL-60 cells (granulocytes) and peripheral blood leukocytes (PBLs). Six of the 55 serum samples were from unvaccinated adult volunteers, 31 serum samples were from adults who received one dose of the 14-valent or the 23-valent polysaccharide vaccine, and 18 serum samples were from 16-month-old infants who received four doses of an investigational 7-valent polysaccharide-protein conjugate vaccine. The results of an opsonophagocytic assay with HL-60 cells correlated highly with those of an assay with PBLs as effector cells (median r for seven serotypes = 0.87: P < 0.01). Opsonophagocytic titers were compared with the immunoglobulin G antibody concentrations determined by enzyme-linked immunosorbent assay (ELISA). The r values for serogroups or serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were 0.61, 0.60, 0.67 0.90, 0.61, 0.39, and 0.57, respectively, when HL-60 cells were used as effector cells and 0.56, 0.47, 0.61, 0.90, 0.71, 0.31, and 0.62, respectively, when PBLs were used. The assay requires small amounts of serum (40 microliters per serotype), making this test suitable for assaying infant sera. Culturable cells aid in assay standardization and likely reduce donor-to-donor variability. This standardized assay, in combination with the standardized ELISA, can be used to evaluate current and developing pneumococcal vaccines, in which functional opsonophagocytic antibody activity may correlate with protection against pneumococcal disease.

Abstract

Exposure to ozone at levels near the National Ambient Air Quality Standard causes respiratory symptoms, changes in lung function, and airway inflammation. Although ozone-induced changes in lung function have been well characterized in healthy individuals, the relationship between airway inflammation and changes in pulmonary function have not been prospectively examined. The purpose of this study was to determine whether individuals who differ in, lung function responsiveness to ozone also differ in susceptibility to airway inflammation and injury. A secondary goal was to determine whether ozone exposure induces airway inflammation in smokers, a population known to have airway inflammation and an increased burden of toxic oxygen species. Healthy nonsmokers (n = 56) and smokers (n = 34) were exposed to 0.22 parts per million (ppm)* ozone for 4 hours, with intermittent exercise, for the purpose of selecting ozone "responders" (decrement in forced expiratory volume in 1 second [FEV1] > 15%) and "nonresponders" (decrement in FEV1 < 5%). Selected subjects then were exposed twice to ozone (0.22 ppm for 4 hours with exercise) and once to air (with the same exposure protocol), each pair of exposures separated by at least 3 weeks, in a randomized, double-blind fashion. Nasal lavage (NL) and bronchoalveolar lavage (BAL) were performed immediately after one ozone exposure and 18 hours after the other, and either immediately or 18 hours after the air exposure. Indicators of airway effects in lavage fluid included changes in inflammatory cells, proinflammatory cytokines, protein markers of epithelial injury and repair, and generation of toxic oxygen species. In the classification exposure, fewer smokers than nonsmokers were responsive to ozone (11.8% vs. 28.6%, respectively); an insufficient number of smoker-responders were identified to study as a separate group. In the BAL study, all groups developed a similar degree of airway inflammation, consisting of increases in interleukins 6 and 8 (maximal immediately after exposure), and increases in polymorphonuclear leukocytes (PMNs), lymphocytes, and mast cells (maximal 18 hours after exposure). The increase in PMNs was inversely correlated with age (p = 0.013), but gender, nonspecific airway responsiveness, and allergy history were not predictive of inflammation. Alveolar macrophage production of toxic oxygen species decreased after ozone exposure in nonsmokers; however, not in smokers. Findings from nasal lavage did not mirror lower airway inflammatory responses in these studies. We conclude that, in response to ozone exposure, smokers experienced smaller decrements in lung function and fewer symptoms than nonsmokers; however, the intensity of the airway inflammatory response was independent of smoking status or airway responsiveness to ozone. Furthermore, the burden of toxic oxygen species following ozone exposure was greater for smokers than for nonsmokers. Subjects were young, healthy, and able to sustain exercise; the results may not be representative of nonsmokers or smokers in general. Nevertheless, the findings indicate that measuring symptoms and spirometric changes is not sufficient to assess the potential risks associated with ozone exposure.

Abstract

As part of pharmacologic studies of asparaginase (ASNase), we determined the half-life of ASNase activity and protein, and the effect of dose, repeated doses, different drug preparations, and hypersensitivity reactions on the half-life (t1/2) of serum ASNase activity.We measured ASNase activity (spectrophotometric assay) in serum samples obtained from patients with acute lymphoblastic leukemia (ALL) at various times during their therapy with intramuscular ASNase. ASNase protein was measured by enzyme-linked immunoadsorbent assay (ELISA).Studies following the initial dose of Escherichia coli-derived ASNase demonstrated no difference in apparent t1/2 following 25,000 IU/m2 versus 2,500 IU/m2 (1.24 v 1.35 days, P = .2). The apparent t1/2s following maintenance doses of E coli ASNase (middle dose t1/2, 1.28 days, or last dose t1/2, 1.14 days) showed no difference when compared with the initial dose of ASNase (P = .3 to .9). There was no significant difference between the apparent t1/2s of ASNase activity and ASNase protein (n = 8, P = .2 to .6). The serum t1/2 was 0.65 and 5.73 days for patients receiving Erwinia or polyethylene glycol (PEG)-modified E coli ASNase, respectively, as the induction dose. ASNase activity was undetectable in sera of four patients studied in the week following an anaphylactic reaction to E coli ASNase and the t1/2 was significantly shorter in five patients with a history of allergic reaction to E coli ASNase who were studied following a dose of PEG ASNase, (t1/2, 1.80 days).We conclude that (1) the apparent t1/2 of ASNase is dependent on enzyme preparation used, but is not affected by dose or by repeated use; (2) the apparent t1/2 of E coli ASNase as a protein is the same as the apparent t1/2 of enzymatic activity; and (3) patients who have had a hypersensitivity reaction to E coli ASNase have a decreased apparent t1/2 with both E coli and PEG ASNase.

Abstract

Treatment of tumor cells with hydroxyurea and other DNA-damaging agents has been shown to increase the experimental metastatic potential of these cells.We sought to elucidate some of the biochemical and genetic changes that promote tumor cell metastasis in hydroxyurea-treated cells. We hypothesized that drug treatment induces resistance to oxidative damage and that elimination of this resistance reverses the drug-induced experimental metastatic capabilities of tumor cells.We examined the effect of hydroxyurea treatment on B16 melanoma cells with respect to experimental metastatic potential, resistance to hydrogen peroxide (H2O2), glutathione peroxidase activity and messenger RNA (mRNA) level, glutathione reductase activity, glutathione levels, glutathione-S-transferase activity, and catalase activity and mRNA level.Hydroxyurea-treated cells were transiently more metastatic following intravenous injection in syngeneic mice and transiently more resistant than untreated cells to exogenous H2O2. Hydroxyurea-induced experimental metastases and H2O2 resistance were eliminated by depletion of intracellular glutathione with buthionine sulfoximine. Glutathione peroxidase activity and mRNA level, glutathione reductase activity, and reduced glutathione levels were all transiently increased in hydroxyurea-treated cells, whereas the increase in glutathione-S-transferase activity was sustained. Catalase activity was modestly increased with no increase in its mRNA levels.In B16 melanoma cells, experimental metastasis induced by hydroxyurea appears to depend on a process that requires glutathione. Hydroxyurea treatment also induces resistance to exogenous H2O2, which may be due to induction of glutathione and antioxidant enzyme activity.The role of antioxidants in B16 melanoma cells offers new insights into the metastatic process and the cellular response to chemotherapy.

Abstract

The effects of a beta-adrenergic agonist and a cyclic AMP analogue on activation, activity, and termination of FMLP-stimulated superoxide anion production were investigated. Incubation with isoproterenol resulted in a 50% reduction in the maximal rate of superoxide production and a 3-4-fold increase in the rate of termination of superoxide production. Exposure to 1 mM dibutyryl cyclic AMP resulted in a 40% decrease in the maximal rate and a 3-fold increase in the rate of termination of FMLP-induced superoxide production. Neither agent had a significant effect on the lag time prior to superoxide anion generation.

Abstract

To determine if prophylactic corticosteroids would prevent acute lung injury caused by hyperoxia and barotrauma, 29 piglets (1.2 +/- 0.3 kg, 1-2 days of age) were studied. Ten piglets were hyperventilated [arterial PCO2 (PaCO2) 15-20 Torr] with 100% O2 for 48 h and compared with 10 piglets treated with the identical management but given 0.7 mg/kg of dexamethasone at time 0 and every 12 h for the 48-h study. Six piglets were normally ventilated (PaCO2 40-45 Torr) for 48 h with 21% O2 as an additional control group. Pulmonary function and tracheal aspirates were examined at time 0 and every 24 h. Bronchoalveolar lavage was performed for surfactant analyses at the conclusion of the study. In animals treated with hyperoxia and hyperventilation, lung compliance decreased 32% and tracheal aspirate polymorphonuclear leukocyte (PMN) chemotactic activity increased by 51%, cell counts by 204%, number of PMNs by 277%, elastase activity by 111%, and albumin concentration by 328% over 48 h (P less than 0.05). In contrast, dexamethasone-treated piglets had increases in only tracheal aspirate albumin concentration (206%) over the 48-h study. All cellular and biochemical variables were lower in dexamethasone-treated compared with hyperoxic hyperventilated piglets. Room air normal ventilation controls had only a 108% increase in tracheal aspirate albumin concentration noted. Despite quantitative differences in surfactant among the three groups, activity was unaffected. Results indicate that hyperoxia and hyperventilation for 48 h causes significant inflammatory changes and acute lung injury and that prophylactic high-dose dexamethasone significantly ameliorates this lung damage.

Abstract

The antileukemic activity of L-asparaginase (ASNase), an important component of therapy for acute lymphoblastic leukemia, is thought to result from depletion of serum L-asparagine (Asn). In studies of the pharmacological effects of ASNase, investigators have reported prolonged reduction in the serum concentration of Asn after the administration of ASNase. Such measurements may not be valid because ASNase present in the blood sample may hydrolyze Asn before its determination. We examined recovery of [U-14C]Asn from blood samples with and without various concentrations of added ASNase. In the presence of greater than or equal to 0.01 IU/ml of ASNase, the amount of [U-14C]Asn recovered was less than 15% of that without ASNase. Utilizing this assay, we studied the effect of 2 known inhibitors of ASNase in an attempt to improve Asn recovery. In the presence of aspartic beta semialdehyde (ASA), or 5-diazo-4-oxo-L-norvaline (DONV), and up to 1.0 IU/ml ASNase, Asn levels remained at greater than 90% of control. ASA prevented the hydrolysis of exogenous Asn in blood samples drawn from patients after ASNase injection. We also developed a method to determine Asn in serum utilizing high pressure liquid chromatography. Using this method, we found that the Asn level was greater than 90% of a normal level in the presence of 40 mM DONV and 1.0 IU/ml ASNase. Examination of serum from 4 patients treated with ASNase showed that Asn is detectable 7-19 days sooner when DONV is present in the blood collection system than in its absence. We conclude that: (a) as little as 0.01 IU/ml ASNase can hydrolyze Asn added to blood; (b) continued hydrolysis of Asn by ASNase ex vivo can result in falsely low serum Asn measurements; (c) ASA or DONV present in the collection tubes obviates the problem of continued ASNase activity; and (d) the degree and duration of Asn depletion after ASNase therapy is much less than previously believed. Thus, for accurate measurements of the duration and degree of Asn depletion by ASNase, an ASNase inhibitor such as ASA or DONV should be present in the blood collection system.

Abstract

Changes in intracellular ionized free calcium ([Ca]i), inositol triphosphate (IP3), and sn-1,2-diacylglycerol (DAG) were determined in relation to agonist-induced human neutrophil superoxide (O2-) production. With 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation, generation of IP3 and a peak rise in [Cai] occurred at 30 sec, preceding maximal O2- production (1.5 min) and the maximal rise in DAG mass (4 min). FMLP-induced O2- production was inhibited by pertussis toxin. In cytochalasin B-primed, concanavalin A (Con A) stimulated neutrophils, a peak rise in [Ca]i but not IP3 proceeded O2- production, and pertussis toxin did not inhibit O2- production. EGTA inhibited the cytochalasin B/fMLP-induced increment in [Ca]i and O2- production by 75% and 50%, respectively, and completely ablated the response to cytochalasin B/Con A, suggesting a role for extracellular as well as intracellular calcium in the respiratory burst. However, three types of experiments indicate that an increase in [Ca]i is neither sufficient nor always required for O2- production. First, treatment with ionomycin resulted in a marked increase in [Ca]i but did not cause O2- production. Second, pertussis toxin inhibited both fMLP-induced IP3 generation and O2- production but did not inhibit the rise in [Ca]i. Third, following neutrophil priming with dioctanoylglycerol (diC8), maximal O2- production occurred in response to 0.015 microM fMLP or Con A without a rise in [Ca]i, and diC8/fMLP-induced O2- production was not inhibited by EGTA. Taken together, these data suggest that 1) an increment in [Ca]i is not strictly essential for neutrophil O2- production, 2) unlike fMLP, Con A-induced O2- production does not proceed through a pathway involving the pertussis toxin-sensitive G protein, and 3) regulation of neutrophil [Ca]i involves mechanisms independent of IP3 concentration.

Abstract

Human plasma glutathione peroxidase (GSHPx) has been shown to be a glycosylated selenoprotein distinct enzymatically, structurally, and antigenically from known cellular glutathione peroxidases. The extracellular location of the enzyme and the fact that it is glycosylated suggested that it is a secreted protein. Utilizing mutually non-cross-reactive antibodies to human cellular and plasma GSHPx, we conducted a search to determine the tissue of origin for plasma GSHPx. The cells screened were endothelial cells because they are the main source of extracellular superoxide dismutase, HL-60 cells (myeloid cell line) because they are the main source of extracellular H2O2, and Hep G2 cells (hepatic cell line) because they are the source of many plasma proteins. Human umbilical vein endothelial cells were metabolically labeled with either [35S]methionine or [75Se]selenious acid, and HL-60 cells and Hep G2 cells were metabolically labeled with [75Se]selenious acid. Proteins were immunopurified from the labeled cells and their media with either anti-red blood cell (RBC) GSHPx IgG or with anti-plasma GSHPx IgG. Utilizing anti-RBC GSHPx IgG, only the cellular form of the enzyme was precipitated from all the cells tested but not from their media. When anti-plasma GSHPx IgG was applied to the cells and their media, a selenoprotein was precipitated only from the media of Hep G2 cells. When Hep G2 cells were incubated in the presence of the carboxylic ionophore monensin, an intracellular selenoprotein could be detected using anti-plasma GSHPx IgG. The precipitation of the cellular form from all three cell types was partially inhibited by preincubation of the anti-RBC GSHPx IgG with purified RBC GSHPx while the precipitation of the selenoprotein from the medium of Hep G2 cells by anti-plasma GSHPx IgG was prevented by preincubation of the antibody with purified plasma GSHPx. We suggest that plasma GSHPx is synthesized by and secreted from hepatic cells. This is, to the best of our knowledge, the only known selenoprotein with a defined function that has been shown to be synthesized for secretion by mammalian cells.

Abstract

Plasma glutathione peroxidase (GSHPx) (glutathione: H2O2 oxidoreductase) is a unique selenoglycoprotein. Treatment of this enzyme with glycopeptidase F partially deglycosylates it and establishes the presence of N-linked sugar moieties. Antibodies raised in a rabbit against the purified enzyme from plasma were found to be specific, noninhibitory, and capable of precipitating the enzymatic activity. The antibodies precipitated greater than 90% of the GSHPx activity of normal plasma, thus indicating that the selenoenzyme is the main if not the sole GSHPx activity of plasma. The antibodies did not precipitate RBC GSHPx. A slight cross-reactivity of the antibodies was found with rat plasma GSHPx. A GSHPx activity precipitation assay of normal plasma in the presence of selenium (Se)-deficient plasma indicates that no cross-reactive protein in the Se-deficient plasma interferes with the precipitation of the GSHPx activity from normal plasma. Thus, GSHPx protein as well as activity is deficient in plasma in the absence of Se. Antibodies against GSHPx either from RBCs or from plasma were used to specifically immunoprecipitate most of the GSHPx activity from RBCs or plasma, respectively, in healthy individuals to determine the amount of Se associated with the protein. GSHPx accounts for approximately 15% of the Se in RBCs and 12% of the Se in plasma. Thus, in normal individuals, these proteins account for only a fraction of plasma and RBC Se.

Abstract

The biochemical mechanisms for the termination of the respiratory burst are likely to be multifactorial. Alterations in the endogenous oxidant-scavenging mechanisms can modulate the kinetics of the termination phase of neutrophil superoxide production. These are most apparent when those alterations are found in the neutrophils of certain patients. The use of inhibitors of the oxidant scavenging systems in normal neutrophils has not yielded results that exactly mimic the studies with neutrophils from the patients. For example, the glutathione reductase-inhibited neutrophils (from the action of BCNU) do not show the same degree of abnormality as do the neutrophils from the patient with glutathione reductase deficiency. Further investigations on the mechanisms of inactivation of the NADPH oxidase are warranted in order to gain a greater understanding of this important regulating mechanism.

Abstract

Glutathione peroxidase (GSHPx), (glutathione:H2O2 oxidoreductase, EC 1.11.1.9) was purified to homogeneity from human plasma. This resulted in a 6800-fold purification of the enzyme with a 2.8% yield. The purification process involved ammonium sulfate fractionation, DEAE-cellulose batch and column chromatographies, hydroxyapatite, and Sephadex G-200 and DEAE-Sephadex A-25 chromatographies. The major peak on DEAE-Sephadex A-25 column chromatography was found to be homogeneous on polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate (SDS). Relative mobility in nondenaturing polyacrylamide gel electrophoresis at pH 8.2 was 0.5 for the purified enzyme as detected by both protein staining and enzyme activity compared with 0.38 for erythrocyte GSHPx. The molecular weight of the plasma enzyme as determined by gel filtration was found to be approximately 100,000. SDS-gel electrophoresis of the plasma enzyme gave a subunit molecular weight of approximately 23,000. This suggests that the plasma enzyme exists as a tetramer in its native state, similar to that seen for the erythrocyte enzyme, but with slightly different mobility on SDS-gel electrophoresis. Plasma GSHPx, like the erythrocyte enzyme, was found to contain approximately four atoms of selenium per mole of protein. Utilizing iodinated concanavalin A, it was found that plasma GSHPx, but not the erythrocyte GSPx, is a glycoprotein. Purified plasma enzyme catalyzes both the reduction of tertiary butyl hydroperoxide and hydrogen peroxide. The apparent Km of plasma GSHPx for GSH is 5.3 mM and for tertiary butyl hydroperoxide it is 0.57 mM. Copper, mercury, and zinc strongly inhibit the enzyme activity of plasma GSHPx. Rabbit antibodies directed against the human erythrocyte GSHPx do not precipitate the enzyme activity of the purified plasma enzyme. Radioimmunoassay utilizing erythrocyte GSHPx and anti-erythrocyte GSHPx antibodies showed that less than 0.13% of the antigenically detectable protein is found in the purified GSHPx from plasma.

Abstract

The effect of concanavalin A (Con A) on the concentration of ionized intracellular calcium [( Cai++]) in human granulocytes (PMN) was monitored using the fluorescent calcium indicator and chelator, Quin2. The addition of Con A to PMN resulted in a rise in [Cai++] that was markedly enhanced by the presence of Ca++ in the external buffer. The onset of the increment in [Cai++] preceded the onset of O2- production. The rise in [Cai++] induced by Con A is not transient, with a new, higher steady-state level of [Cai++] being attained within five minutes. The addition of alpha-methylmannoside (alpha-MM) one minute after Con A resulted in the return of [Cai++] to the original baseline level and the cessation of O2- production. The addition of a second stimulus (such as arachidonic acid) to these cells resulted in a second increment in [Cai++] and the return of O2- production. Thus the rise in [Cai++] induced by Con A is tightly coupled to the activation, inactivation, and reactivation of the O2- generating system by Con A. Further experiments were undertaken to assess the possible requirement for the rise in [Cai++] in the activation of PMN by Con A. PMN could be depleted of Cai++ by loading with Quin2 in the absence of extracellular Ca++. These Ca++- depleted PMN can be induced to produce O2- after treatment with PMA but not with Con A. The addition of Ca++ to Ca++ -depleted PMN results in a return of [Cai++] to the normal resting level of Ca++ -replete PMN. The time required to return to baseline is a function of the concentration of intracellular Quin2. The addition of Ca++ to Ca++ -depleted, Con A-treated PMN results in the elevation of [Cai++] and the production of O2-. Over a tenfold range of intracellular Quin2 concentration, the onset of O2- production always occurred at [Cai++] that were less than the normal resting level. Thus, activation of the O2- generating system by Con A can occur at [Cai++] which are much lower than the incremental level induced by Con A in Ca++ -replete PMN. Supporting this is the observation that only a very small increment in [Cai++] is induced by Con A in PMN cytoplasts, even though Con A could induce O2- production in the Quin2-loaded cytoplasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract

Human granulocytes (polymorphonuclear leukocytes, PMN) produce H2O2 and other reactive oxygen species while undergoing phagocytosis. To examine the role of the glutathione cycle in metabolizing H2O2, we incubated PMN with 1,3-bis (2-chloroethyl) nitrosourea (BCNU). Incubation of PMN with BCNU results in a dose-dependent inhibition of PMN glutathione reductase (GRED), with 50% inhibition occurring at approximately 2 micrograms/mL BCNU. PMN hexose monophosphate shunt activity stimulated with an exogenous H2O2-generating system was inhibited only when the GRED activity was reduced to less than 30% of control. BCNU-treated cells contained lower levels of reduced sulfhydryls and reduced glutathione, which decreased even more in the presence of an exogenous H2O2-generating system. The effect of BCNU and exogenous H2O2 on various aspects of phagocytosis were examined. Exposure of BCNU-treated PMN to an H2O2-generating system resulted in an inhibition of chemotactic peptide-induced shape changes and degranulation. The ability of BCNU-treated cells to produce O2- was diminished only when the PMN were incubated with an H2O2-generating system in the presence of cyanide. Ingestion of opsonized bacteria by BCNU-treated PMN was unaffected by incubation in an H2O2-generating system even in the presence of cyanide. We conclude that PMN GRED is inhibited by BCNU, the ability of PMN to metabolize H2O2 is affected only when GRED is reduced more than 70%, this inhibition affects the glutathione content of these cells, and some, but not all of the phagocytic functions of GRED-inhibited PMN are inhibited after exposure to an H2O2-generating system.

Abstract

B cell lymphomas which can be growth inhibited by crosslinking their surface IgM receptors by anti-Ig reagents provide models for normal B cell regulation and tolerance. WEHI-231 and CH31 are two independently derived lines that are exquisitely sensitive to negative signalling by antibodies specific for mu or kappa chains, but are unaffected by antibodies against MHC class 1 or 2 antigens. In order to determine the mechanism of this growth inhibition as a model for tolerance, we have examined the roles played by protein kinase C activation and calcium mobilization/influx during negative signalling in these cells. We found that growth inhibition caused by anti-mu crosslinking was reversed in the presence of either phorbol myristate acetate (PMA) or by lipopolysaccharide (LPS) from E. coli. The effect of PMA on negative signalling was a true reversal since phorbol esters could be added after anti-mu treatment, thus allowing nearly normal cellular progression into the S phase of the cell cycle. In contrast, pretreatment with PMA did not provide protection against the growth inhibition from anti-mu. Indeed, a "desensitization" protocol demonstrated that PMA pretreatment actually decreased reversal by both PMA and LPS of the effects of anti-mu on B lymphoma growth. These studies suggest that both LPS and PMA act via at least one common intermediate, which is assumed to involve activation and translocation of protein kinase C. Analysis of changes in calcium ion concentration after treatment with anti-Ig reagents showed both mobilization from internal stores and influx via calcium channels in WEHI-231, as has been reported for normal B cells. However, these changes did not correlate with negative signalling for the several reasons. Firstly, anti-mu inhibition of the growth of WEHI-231 could be induced in the relative absence of extracellular Ca++ or in quin-2 loaded (buffered) cells. Secondly, pretreatment with high concentrations of PMA ablated calcium mobilization, yet failed to modulate growth inhibition in WEHI-231 cells. Moreover, LPS provided protection from the effects of anti-mu yet did not alter cellular [Cai++]. In addition, PMA posttreatment (under conditions causing a reversal of the effects of anti-mu) can be applied as long as four hours after the initial exposure to anti-mu and the rapid measurable changes in calcium flux. Indeed, such changes in intracellular free calcium occurred in elutriated WEHI-231 lymphoma cells at all phases of the cell cycle, although we have previously identified early G1 as the only critical period in which negative signalling can be delivered.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract

We have utilized monoclonal antibodies against the two IgG Fc receptors (p40 and p72) of U937 cells to stimulate the release of superoxide. The monoclonal antibody (mAb) specific for p40 (IV3) has been described elsewhere. A murine IgG1 mAb specific for the high affinity p72 Fc receptor (designated mAb FcR32 or simply mAb 32) bound to the same p72 precipitated by Sepharose-human IgG as shown by preclearing experiments and by identical isoelectric focussing patterns. Binding of mAb 32 to p72 was independent of the Fc region of the antibody since Fab' fragments of mAb 32 affinity adsorbed p72. The binding of both mAb 32 and human IgG1 to the intact U937 cell was not reciprocally inhibitory, indicating that mAb 32 does not interfere with the ligand binding site of p72. mAb 32 bound to human monocytes, U937, and HL60 cells, but not to granulocytes or lymphocytes. U937 cells cultured in gamma-interferon and 1,25-dihydroxycholecalciferol generated superoxide when incubated with mAb 32 or IV3 followed by cross-linking with F(ab')2 anti-murine Ig. Incubation with mAb 32 or IV3 alone or with 3 of 5 other anti-U937 mAbs cross-linked with anti-murine Ig did not result in superoxide generation. Immune complex-mediated superoxide production was inhibited 80% by IgG, but not by mAb 32 or IV3.

Abstract

The effects of arachidonic acid on human granulocytes (polymorphonuclear neutrophil leukocytes, PMN) were examined with respect to early events associated with activation of the superoxide (O2-)-generating system and its reactivation with other stimuli after the addition of fatty acid-free bovine serum albumin. As with other stimuli, O2- production occurred after a lag that was dependent on the amount of arachidonic acid used. Although the lag, rate, and extent of O2- production were dose dependent, at all concentrations used, the duration of O2- production was four to six minutes. As previously described, when fatty acid-free albumin was added one minute after stimulation of PMN by arachidonic acid, O2- production ceased immediately. The O2(-)-generating system could then be reactivated by the addition of either phorbol myristate acetate (PMA), concanavalin A, or serum-treated zymosan. In previously activated cells, the lag time for reactivation was shorter and the rate of O2- production greater with PMA. PMN membrane potential was depolarized by arachidonic acid. This depolarization was not reversed with the addition of albumin. PMN cytoplasts were also capable of reversible activation by arachidonic acid in a manner identical to whole cells. Divalent cations were found to be necessary for the activation of PMN by arachidonic acid. In the absence of calcium, arachidonic acid caused rapid lysis of the cells, as manifested by the release of lactic acid dehydrogenase. We were unable to measure later events in PMN stimulated by arachidonic acid since even in the presence of divalent cations lactic acid dehydrogenase was released from the cells after five minutes of incubation. We conclude that, in addition to its ability to activate PMN, arachidonic acid is toxic to the cells and cannot be used to study late events in granulocyte activity, and, in the absence of calcium, it may be difficult to interpret its action as a stimulant of the oxidase.

Abstract

Incubation of platelets with pronase or chymotrypsin results in the exposure of fibrinogen receptors. We determined that these enzymes did not affect the membrane fluidity as evaluated by the depolarization of the fluorescence of 1,6-diphenyl-1,3,5 hexatriene (DPH). There was no significant difference in either the depolarization or in its temperature dependence for control, pronase or chymotrypsin-treated platelets. Thus, it can be concluded that the exposure of fibrinogen receptors on the platelet surface by proteolytic enzymes does not depend on the changes of membrane fluidity. We also propose that the proteolytic enzymes do not cause a major alteration in the extent of protein chains embedded in the lipid layers of the platelet membranes.

Abstract

Aggregation and the activation of the granulocyte (PMN) superoxide (O2-) generating system occur when certain stimuli are added to resting cells. It had previously been postulated that PMN aggregation is essential for maximal O2- production. This study was undertaken to test the hypothesis that PMN aggregation is required for full expression of PMN O2- production. We examined aggregation and O2- production induced by four stimuli; concanavalin A (Con A), phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), and ionophore A23187. Cytochalasin B enhanced aggregation by all four stimuli but only enhanced the rate of O2- production by Con A; 2-deoxyglucose inhibited aggregation by all stimuli. Dissociation of PMN aggregation and O2- production was achieved by using NEM, TPCK, and divalent cations. NEM and TPCK prevent Con A-induced O2- production but have no effect on Con A-induced aggregation. PMA-stimulated PMN generate O2- in the presence or absence of Ca++ and Mg++. In contrast, PMA stimulated maximum PMN aggregation only in the presence of both Ca++ and Mg++. Thus PMN can generate O2- without aggregating, and PMN can aggregate without producing O2-. PMN from patients with chronic granulomatous disease do not generate O2- or undergo membrane potential depolarization in response to PMA. These PMN aggregated when stimulated with PMA, providing evidence that depolarization is not required for PMN aggregation. We conclude that aggregation and the activation of the O2- generating system, though temporally related, are not necessarily causally related.

Abstract

A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta-glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3'-dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor-mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses.

Abstract

Utilizing the induced differentiation of HL-60 promyelocytic leukemia cells as a model of myeloid maturation, we examined the development of the superoxide-generating system, focusing on NADPH oxidase activity, membrane depolarization, and cytochrome b content. NADPH oxidase activity, measured as NADPH-dependent superoxide production, increased with both spontaneous and N,N-dimethylformamide-induced differentiation. Activity in particulate fractions from induced HL-60 cells and human peripheral blood polymorphonuclear leukocytes was proportional to their relative rates of superoxide production, but activity from uninduced cells was surprisingly high: one-third that from induced cells, despite only 7% their rate of superoxide generation. NADPH oxidase activities in phagocytic vesicles from induced HL-60 cells and polymorphonuclear leukocytes were equal, indicating the equivalence of the enzyme system in active portions of their cell membranes. Separation by centrifugal elutriation of the HL-60 cell population into fractions of varying maturity confirmed the relationship of NADPH oxidase activity to advancing differentiation in both dimethylformamide-induced and spontaneously maturing cells. Membrane potential change, an early event related to activation of the oxidase, was followed by 3,3'-dipropylthiodicarbocyanine dye fluorescence. The depolarization response increased dramatically in both magnitude and initial rate of change during differentiation. The cells' cytochrome b content increased 3-fold with induction of differentiation, in proportion to the change in NADPH oxidase activity.

Abstract

The stimulation of granulocyte O2- production by concanavalin-A can be reversed with alpha-methylmannoside. Such cells can be reactivated to generate O2- by adding phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine. Opsonized zymosan, however, is not an effective stimulant to these cells. alpha-Methylmannoside prevents, but does not reverse, depolarization of granulocytes by concanavalin-A. Previously activated cells have a shorter lag time for reactivation by phorbol myristate acetate. Incubation in 2-deoxyglucose of cells previously treated with concanavalin-A and alpha-methylmannoside prevents reactivation. EGTA prevents concanavalin-A-stimulated O2- production only when added prior to the stimulant. EGTA has only a slight effect on reactivation. alpha-Methylmannoside prevents concanavalin-A-stimulated release of lysozyme only when added prior to the stimulant. Prior treatment of cells with concanavalin-A and alpha-methylmannoside inhibits subsequent ingestion of complement-coated particles. We conclude that although the O2--generating system can be reversibly activated with concanavalin-A followed by alpha-methylmannoside, these cells are different from untreated cells. Cells treated in such a way do not respond to all stimuli, remain depolarized, have shortened lag times, no longer require calcium for activation, continue to degranulate, and do not ingest well. Thus, although some changes that accompany the interaction of stimuli with granulocytes are reversible, some are not, and the previously activated cell does not return to a true resting state.

Abstract

A 7-yr-old girl with self-limited pulmonary aspergillosis was found to have a defect in granulocyte superoxide production. Her cells produced superoxide at 3% of control rates in response to phorbol myristate acetate (PMA) and opsonized zymosan. Lag times for O-2 production were normal with PMA, opsonized zymosan, and concanavalin-A stimulation. Her granulocyte membranes depolarized in response to all of these stimuli. Superoxide produced by podosomes and a particulate fraction demonstrated an enzyme activity with a normal maximal velocity but a decreased affinity for NADPH. NADH-dependent superoxide production by particles was similar with patient and control material. The duration of superoxide production was prolonged in the patient's intact granulocytes and in the particulate fractions from her cells. Bacterial killing by the patient's granulocytes was initially low, but approached control values after 90 min of incubation. These results are explained by an enzyme activity that has a decreased affinity for its substrate and a decreased rate of inactivation. Family studies indicate an autosomal recessive mode of inheritance.

Abstract

The effects of alpha-tocopherol (vitamin E) deficiency on membrane properties of platelets were studied to determine if vitamin E has a measureable stabilizing role in biological membranes. Three groups of rats and three of mice were studied: two groups consisted of Fisher strain rats and one of Sprague-Dawley rats fed a Draper corn oil diet with and without high levels of supplementary vitamin E. The mice were two groups of BALB/c animals maintained on an 8% hydrogenated coconut oil diet, and one group of CBA/J mice on an 8% lard diet, in each case either deficient in or supplemented with vitamin E. The relative content of fatty acids obtained from both rat platelets and erythrocytes was unchanged by vitamin E deficiency. Depletion of vitamin E had no effect on the degree of fluorescence polarization of 1,6-diphenyl-1,3,5,-hexatriene-labeled rat platelets. No changes in hematocrit values were seen in any of the studies. The platelet count of only the vitamin E-deficient Sprague-Dawley rats was elevated with respect to vitamin E-supplemented counterparts; the others remained constant. Platelet reactivities, as measured by ADP-and thrombin-induced platelet aggregation and by the thrombin-induced changes in platelet transmembrane potential, were unaffected by vitamin E deficiency in all three groups of rats. Our results indicate that a membrane stabilizing effect of vitamin E on rat platelet or erythrocyte membrane fatty acids or on platelet response to external stimuli could not be demonstrated, nor was elevation in platelet count a general phenomenon associated with vitamin E deficiency.-Whitin, J. C., R. K. Gordon, L. M. Corwin, and E. R. Simons. The effect of vitamin E deficiency on some platelet membrane properties.

Abstract

Phagocytic cells generate superoxide in response to stimulation by opsonized particles. A continuous assay for opsonized zymosan-stimulated granulocyte superoxide production shows that there is a lag time between the addition of particles and the onset of detectable superoxide production. Superoxide production is preceded by membrane potential depolarization. Neither superoxide production nor membrane depolarization occurs in granulocytes from patients with chronic granulomatous disease. The extent of activation by opsonized zymosan is affected by the dose of zymosan from 0.5 to 4.5 mg/ml, but the time necessary for activation (lag time) is not. Similarly, the extent of depolarization but not the time necessary for attaining maximum depolarization is concentration-dependent. Effects of temperature, divalent cations, 2-deoxyglucose, cyanide, and N-ethyl maleimide on superoxide production are similar for granulocytes treated with soluble stimuli and with opsonized zymosan. Thus, zymosan stimulates granulocytes to generate superoxide and undergo membrane depolarization in a manner similar to that elected by soluble stimuli.

Abstract

Activation of normal or myeloperoxidase-deficient human granulocytes by phorbol myristate acetate resulted in an initial membrane depolarization as indicated by an increase in fluorescence of the lipophilic cation probe of membrane potential, 3,3'-dipropylthiodicarbocyanine. A subsequent apparent hyperpolarization (decrease in fluorescence) was observed in normal but not myeloperoxidase-deficient cells. Addition of purified myeloperoxidase restored a normal pattern of fluorescence changes to the enzyme-deficient granulocytes. The secondary decrease in fluorescence in normal cells was markedly blunted by addition of azide, cyanide, or catalase. In a cell-free system, the fluorescence of 3,3'-dipropylthiodicarbocyanine, but not that of 3,3'-dipentyloxadicarbocyanine, was rapidly eliminated by myeloperoxidase in the presence of hydrogen peroxide and a halide; this loss of fluorescence was inhibited by azide, cyanide, or catalase. These findings indicate that secretion of myeloperoxidase and hydrogen peroxide by activated granulocytes results in decreased fluorescence of 3,3'-dipropylthiodicarbocyanine, probably by thioether oxidation. While the determination of initial rates of depolarization using this probe is unaffected by the myeloperoxidase system, measurement of extent of depolarization and any subsequent membrane potential changes requires the addition of inhibitors. In the absence of inhibitors, the secondary decrease in fluorescence can be used as an indicator of secretion of myeloperoxidase and hydrogen peroxide.

Abstract

Circular dichroism and difference spectroscopy have been used to study dilute aqueous solutions of oxygenated, deoxygenated, and carbamoylated deoxygenated hemoglobins A and S (HbA and HbS, respectively). The spectra of HbA and HbS, in comparable state of oxygenation or carbamoylation, are identical, strongly implying identical conformations about the heme groups of the respective proteins. The spectra of the oxygenated forms change little upon addition of KCNO, which is known to carbamoylate the NH2 terminals of the individual chains (Cerami and Manning, 1971). The spectra of the deoxygenated forms, on the other hand, are markedly altered. The decreased magnitude of the 430-nm extremum with increased cyanate concentration can be used to calculate an addition curve which becomes asymptotic at a cyanate:heme molar ratio of approximately 10(3). This conformational change occurs in the absence of O2 and has been predicted (Njikam et al.,1973); it can also be demonstrated by difference spectroscopy techniques, whereby a comparable addition curve can be constructed from changes in the 555-nm absorption, while the 541-nm absorption remains invariant. The change described corresponds to the formation of a new conformation, corresponding to carbamoyldeoxyhemoglobin, carrying one carbamoyl group per chain. In the presence of a small quantity of oxygen, however, the above reported changes in CD are accompanied by a concomitant rise in the 415-nm peak-corresponding to the formation of oxyhemoglobin-while those in the difference spectra reflect not only a change in the 555-nm band but also a parallel one at 541 nm, confirming the formation of oxyhemoglobin. Thus the conformation achieved upon carbamoylation of deoxyhemoglobin has the higher oxygen affinity predicted by Nigen et al. (1974) for carbamoyldeoxyhemoglobin. Cyanate has been used (Cerami and Manning, 1971) as an antisickling reagent in vivo and in vitro, but, although it has been shown that it binds covalently to the NH2-terminal residues of hemoglobin (Lee and Manning, 1973), its effect on hemoglobin conformation has not been previously shown nor has its mechanism of action been fully clarified. The results presented here show that the effect of cyanate on hemoglobin is the formation of a new conformation with heightened oxygen affinity. Since oxyHbS does not aggregate while deoxyHbS does, in a temperature-dependent fashion, the formation of carbamoyldeoxyHbS interferes with such aggregation in vitro in deoxygenated samples. In vivo, where there are generally low residual concentrations of O2, the formation of oxyHb is favored by the higher O2 affinity of carbamoyldeoxyHbS, and aggregation with concomitant red cell sickling is therefore disfavored.